Despite the wealth of structural, chemical and physiological information available, no wholly satisfactory model has yet emerged for the molecular basis of respiration, the transport of oxygen and carbon dioxide by hemoglobin. In particular, the nature of the structural changes which take place on binding a release of oxygen, and the order in which they occur, is still a matter of controversy. The Hoard-Perutz model for the initial conformational "trigger" involves subtle changes in heme stereochemistry on binding ligands. These changes are then transmitted via heme - globin interactions to the interfaces between the chains, ultimately leading to the change in quaternary structure and the expression of cooperativity. The abnormally low affinity of deoxyhemoglobin is ascribed to strain exerted by the globin in the deoxy quaternary structure on the heme, which distorts the iron - porphyrin bonding. This proposal seeks to test the Hoard-Perutz model by preparing horse hemoglobins with chemically modified hemes, both by altering the substituents on the porphyrin ring and by replacing the iron by other metals. Such modifications will probe the heme - globin interactions directly. The alterations in structure will be determined by the x-ray difference Fourier techniques of protein crystallography, and correlated with the alterations in functional properties observed (in collaboration with Professors Gibson, Edelstein and Ho) by such techniques as ligand equilibria, rapid kinetics, ultracentrifugation and proton NMR spectra.